Sulfation of sialic acid is ubiquitous and essential for vertebrate development

Glycosylation of proteins and lipids occurs in vertebrates, usually terminating with sialylation, which regulates the physicochemical and biological properties of these glycoconjugates. Although less commonly known, sialic acid residues also undergo various modifications, such as acetylation, methylation, and sulfation. However, except for acetylation, the enzymes or functions of the other modification processes are unknown. To the best of our knowledge, this study is the first to demonstrate the ubiquitous occurrence of sulfated sialic acids and two genes encoding the sialate: O-sulfotransferases 1 and 2 in vertebrates. These two enzymes showed about 50% amino acid sequence identity, and appeared to be complementary to each other in acceptor substrate preferences. Gene targeting experiments showed that the deficiency of these genes was lethal for medaka fish during young fry development and accompanied by different phenotypes. Thus, the sulfation of sialic acids is essential for the vertebrate development.


Results
Ubiquitous occurrence of SiaS in mammal. First, we investigated the occurrence and distribution of SiaS in various vertebrate cells and tissues by immunodetection with the 3G9 monoclonal antibody, which specifically recognizes 8-O-sulfated N-acetylneuraminic acid (Neu5Ac8S) 45,46 . SiaS was detected in all the examined tissue sections from mice and humans (Fig. 2a, Supp_FigS1), including the kidney, liver, brain, heart, testis, and ovary tissues. Developmental expression of SiaS in the brain was also investigated by western blotting using 3G9 (Fig. 2b). The SiaS epitope was detected at a > 250 kDa smear in fetal brain tissue acquired at 14.5 days postfertilization (E14.5), while < 100 kDa components were increased in neonates (Fig. 2b). SiaS was also chemically detected in the embryonic brain at E14.5 by quantifying the amount of Neu5Ac8S on every slit of the blotted membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorometric high-performance liquid chromatography (HPLC) analysis (Fig. 2c). The amount of Neu5Ac8S was prominent in slit 6 at approximately 65 kDa, although it was also detected in all other slits. The findings indicated that SiaS is expressed in various organs in mice and humans, and in a developmental stage-dependent manner in the mouse brain. The findings further indicated that the ubiquity of SiaS in mammals is much more frequent than has been recognized. cDNA cloning of the sialate: O-sulfotransferase (SulT-Sia). We next sought to clone the gene for an O-sulfotransferase enzyme responsible for transferring the sulfonyl group to the hydroxy group of Sia (SulT-Sia). Based on the two conserved motifs of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-binding domain among sulfotransferases, 61 genes that were already annotated as sulfotransferases in mice were selected (Supp_ DataS1). Although the acceptor substrates for most had already been predicted, two genes appeared to code for sulfotransferases of unknown acceptor substrate specificity: Wscd1 (wall integrity and stress response component [WSC] domain containing 1; NCBI Gene ID: 216881) and Wscd2 (WSC domain containing 2; NCBI Gene ID: 320916). Wscd1 and Wscd2 full-length cDNA was cloned by RT-PCR using total RNA from the E14.5 mouse embryonic brain (Supp_FigS2a). The nucleotide sequences of Wscd1 and Wscd2 contained open reading frames of 1719 bp and 1716 bp, respectively, encoding 572 and 571 amino acid residues (Supp_FigS2b, Fig. 3a). The mouse Wscd1 (mWscd1) and mWscd2 showed 49% identity and shared two conserved PAPS binding motifs: 5'-PSB and 3'-PB (Supp_FigS2c). mWscd1 and mWscd2 belong to a unique clade different from other known glycan-specific sulfotransferases (Supp_FigS3a, Supp_DataS1). Orthologous genes are ubiquitously distributed in the deuterostomes from echinoderms to vertebrates (Supp_FigS3b, Supp_DataS2), which is consistent with the reported occurrence of SiaS in sea urchin [31][32][33][34][35][36][37][38][39] and mammals 40-44 . Identification of Wscd1 and Wscd2 as SulT-Sias. To determine whether Wscd1 and Wscd2 were actually the SulT-Sias, the open reading frames of mWscd1 and mWscd2 were cloned into the pcDNA3.1-V5/His plasmid and used to transfect CHO cells. Forty-eight hours following transfection, both enzymes were detected at 71 kDa by western blotting with anti-V5 antibody (Fig. 3b,e), which coincided with their predicted molecular masses. The cell surface expression of SiaS was analyzed using flow cytometric analysis (FCA) with 3G9 (Fig. 3c,f). An apparent increase in 3G9-positive cells, as well as the % proportion of 3G9-positive cell population of the histograms, were observed for both mWscd1-and mWscd2-transfected cells compared to the mock cells (Fig. 3c,d,f,g). These results suggest that mWscd1 and mWscd2 have SulT-Sia activity. A previous study demonstrated that site-directed mutagenesis of the conserved 5'-PSB motifs (Fig. 3a) abolished sulfotransferase activity 47 . To confirm that mWscd1 and mWscd2 were SulT-Sias, alanine mutants within the 5′-PSB region were constructed (mutWscd1 and mutWscd2, respectively; Supp_FigS4) and expressed in CHO cells to determine the surface expression of SiaS (Fig. 3c,e). FCA revealed a significant decrease in the levels of 3G9 epitope for mut-

Intracellular localization of mWscd1 and mWscd2.
Based on their predicted amino acid sequences ( Fig. 3a), mWscd1 and mWscd2 were identified as type II transmembrane proteins with a short N-terminal cytosolic tail and a C-terminal catalytic domain. V5-tagged mWscd1 and mWscd2 were expressed in CHO cells. They were co-immunostained with GM130 but not KDEL (Fig. 4). Thus, they were Golgi-localized, as predicted.
In vitro activity of mWscd1 and mWscd2. To  www.nature.com/scientificreports/ For the glycolipid substrate, GM1 was incubated with the recombinant mWscd1, mWscd2, and Mock-derived enzyme fraction (Mock), and the reaction products were analyzed by thin-layer chromatography (TLC) (Fig. 5a). The bands denoted by the asterisk were detected for all reaction mixtures where the GM1 substrate was used, consistent with the migration rate of authentic GM1 control. On the other hand, the band denoted by P only appeared in the Wscd1 lane (Fig. 5a), suggesting that the activity of mWscd1 was specific for GM1. No band other than GM1 was detected in the Wscd2 lane (Fig. 5a), suggesting that GM1 was not the substrate of mWscd2. The band P in the Wscd1 lane and the same area in the Mock lane were then extracted for fluorometric HPLC analysis (Fig. 5b). The peak corresponding to the authentic Neu5Ac8S (Fig. 5b authentic) was detected in Wscd1 ( Fig. 5b Wscd1 + GM1), but not in the Mock fractions ( Fig. 5b Mock + GM1). The co-injection experiment with authentic Neu5Ac8S also confirmed that the peak was Neu5Ac8S (Fig. 5b Wscd1 + GM1 + Neu5Ac8S). These results indicate that mWscd1 shows SulT-Sia activity on Neu5Ac residue on GM1, while mWscd2 has no activity against GM1.
The TF glycoprotein substrate, containing two N-glycan chains terminated with α2,6-Neu5Ac residues, was incubated with mWscd1, mWscd2, and Mock, and subjected to western blotting with 3G9 (Fig. 5c). TF was shown to contain the pre-existing 3G9 epitope (Fig. 5c upper lane 8); however, only when incubated with mWscd2 ( Fig. 5c upper lane 5), but not mWscd1 (Fig. 5c upper lane 2) or Mock (Fig. 5c upper lane 1), the 3G9 epitope www.nature.com/scientificreports/ intensity was greatly increased in the TF band at 78 kDa. The amount of TF analyzed in each lane was the same based on the Coomassie Brilliant Blue (CBB) staining intensity of the same gel ( Fig. 5c lower). In addition, this intensity increase did not occur in the absence of PAPS ( Fig. 5c upper lanes 4,6) or TF ( Fig. 5c upper lanes 3, 7).
These results indicate that Wscd2 shows SulT-Sia activity on TF, whereas Wscd1 has no effect on TF. The reaction product of Wscd2 was also analyzed to determine the increase in SiaS by fluorometric HPLC (Fig. 5d). The Neu5Ac8S peak was detected in the reaction product of Wscd2 ( Fig. 5d Wscd2 + TF) but not in Mock ( Fig. 5d Mock + TF). The co-injection experiment also confirmed that the peak was Neu5Ac8S ( Fig. 5d Wscd2 + TF + Neu-5Ac8S). Thus, Wscd2 shows SulT-Sia activity on the Neu5Ac residue of the TF N-glycan.

Characterization of medaka Wscd1 and Wscd2. To gain insight into the significance of Wscd1 and
Wscd2 in vertebrates at the organism level, we chose the medaka fish, Oryzias latipes, as a vertebrate model. Medaka has a single copy of each Wscd1 and Wscd2 orthologs genes. The Wscd1 and Wscd2 cDNAs were cloned from the fry at 6 days post-fertilization (dpf) and 7 dpf, respectively. Their deduced amino acid sequences showed 72% and 75% identity with those from mice, respectively (Supp_FigS6). Based on the real-time quantitative PCR (qRT-PCR) results, these genes were expressed in developing fry at least before hatching (9 dpf), especially after 2 dpf (Supp_FigS7a). They were also ubiquitously expressed in various organs of 3-month-old adult fish (Supp_FigS7b). Both genes were expressed at comparable levels in the kidney, eye, spleen, heart, intestine, and testis tissues, while the expression of Wscd1 was dominant in the brain, liver, muscle, and ovary tissue, the latter being most prominent (Supp_FigS7b). These cDNAs were transfected and expressed in the CHO cells to investigate the SulT-Sia activity. Both enzymes exhibited SulT-Sia activity, based on the results from the FCA with 3G9 (Supp_FigS8). An apparent increase in the % proportion of 3G9-positive cell population of the histograms was observed for both medaka Wscd1-and Wscd2-transfected cells compared to the mock cells.
Although 50% of the Wscd2(−/−) fry died by 18 dpf, 8% lived as long as 60 dpf. Notably, Wscd2(−/−) fry at 50 dpf were smaller than the WT by 49% and 17% in weight and length, respectively (Fig. 6e). Even at 8 dpf, Wscd2(−/−) fry showed smaller eyes and brains than the WT, while Wscd1(−/−) fry did not (Supp_FigS10). www.nature.com/scientificreports/ These observations suggest that growth retardation might occur in Wscd2(−/−) fry, partly due to impaired muscle, eyes and brain development. Interestingly, even heterozygous fry of Wscd1(+/−) and Wscd2(+/−) showed high lethality rates (84% and 43% compared to the WT, respectively; Fig. 6a,b). The remaining fry survived to maturity (approximately 90 dpf), and were fertile to provide the next generation. Considering that the heterozygous fry may express half the amounts of enzymes compared to the WT, the expression levels of Wscd1 and Wscd2 might critically affect the fry survival during the growth stages between 46 and 60 dpf. A notable feature of this period is that the survival curves showed a gradual, but not an acute, reduction (Fig. 6a,b). This result might be related to the severity of the inflammation states in these heterozygous fries. Since medaka fry were reared in non-sterile laboratory conditions, opportunistic infections were possible. Therefore, the inflammation states of fry at 15 dpf were investigated by monitoring the C-reactive protein (CRP) expression level, a marker of inflammation 50 , by qRT-PCR (Supp_FigS11). The CRP expression level was increased in Wscd1(+/−) fry compared to the WT or Wscd1(+/+) fry and was even higher in Wscd1(−/−) fry (Supp_FigS11 left panel). The same or even more prominent results were obtained for Wscd2(+/−) and Wscd2(−/−) fry (Supp_FigS11 right panel). Since CRP increases in response to inflammation 51 , the homozygous and heterozygous fry of both types are suggested to be in an inflammatory state, which is more severe in the homozygous than heterozygous fry. The findings further suggest that the loss of Wscd1 or Wscd2 increases the severity of inflammation in the fry, probably through the dysfunction of various tissues during the growth stages. Further studies are necessary to understand the linkage between the deficiency of these SulT-Sias and the inflamed state. The collective findings indicate the critical roles of Wscd1 and Wscd2 in the survival of medaka.

Discussion
In conclusion, SiaS occurs in various cells and tissues in vertebrates including fish and mammals, and the sialate: O-sulfotransferases, Wscd1 and Wscd2, responsible for the sulfation of Sia residues on glycoproteins and glycolipids are constitutively expressed during embryogenesis and in various adult organs. Wscd1 and Wscd2 are structurally and phylogenetically close to each other, and this gene pair widely distributes in the deuterostomes from echinoderms to vertebrates. Interestingly, mWscd1 and mWscd2 may have complementary substrate www.nature.com/scientificreports/ preferences to each other, because mWscd1, but not Wscd2, is active for glycolipid GM1, while mWscd2, but not mWscd1, for glycoprotein transferrin. This feature might explain constitutive co-expression profiles of these two genes in embryos and adult organs of medaka. Both enzymes are Golgi-localized, type II transmembrane proteins with a short N-terminal cytosolic tail and a C-terminal luminal catalytic domain, and share the common structural and topological features with sialyltransferases 52,53 . Thus, sequential reactions of sialylation and sulfation may effectively proceed in the Golgi compartment. Wscd1 and Wscd2 are the second examples of Sia modification enzymes, following the discovery of sialate: 9-O-acetyltransferase CASD1 29,30 . CASD1 converts CMP-Sia into CMP-Sia9Ac using acetyl-coenzyme A as a donor substrate 30 . Although both Sia modification enzymes are localized in the Golgi, Wscd1 and Wscd2 transfer sulfate group on sialoglycans on proteins and lipids, but not CMP-Sia, using PAPS as a donor substrate (Fig. 7). It may be concluded that Sia modifications occur in the Golgi compartment, although O-sulfation and O-acetylation of Sia residues occur at different metabolite levels before and after sialylation, respectively.
Examination of gene knockout medaka revealed that SiaS is essential at the organism level. Wscd1(−/−), but not Wscd2(−/−), fry at 8 dpf suffer from cardiac arrhythmia 48 , suggesting that Wscd1 is essential for heart development. Wscd2(−/−), but not Wscd1(−/−), fry show growth retardation, accompanying impaired muscle, eyes, and brain development. Wscd2 might be involved in survival of cells in muscle, eye, and brain. Furthermore, the loss of Wscd1 or Wscd2 increases the severity of inflammation in the fry, which may be related to the lethality of fry. Although further studies are necessary to gain in-depth insight into pathophysiology of the lethality, the Sia: O-sulfation must have multiple functions such as embryonic development, organogenesis, and immune recognition. Finally, the present data emphasize the importance of modified Sias that have been uncovered for a long time.
Now that two different genes for SulT-Sia are evident, many questions would immediately emerge in various aspects of biochemistry and biology of SiaS. We can ask if there are still other genes for SulT-Sia. Recently, we reported that the surface expression of SiaS reversibly induced by treatment of CHO cells with the antibiotic G418 54 . Since CHO cells did not express Wscd1 or Wscd2 before and after the G418 treatment (unpublished results), the presence of other SulT-Sia than Wscd1 and Wscd2 is suggested in CHO cells. Thus, more sulfotransferases with the SulT-Sia activity might occur in mammalian cells. We can also seek for substrate specificity of the SulT-Sias in detail. This report shows that Wscd1 and Wscd2 can synthesize Neu5Ac8S-residues from Neu5Ac-residues; however, Sia species-specificity of the enzymes to Neu5Ac, Neu5Gc, and Kdn, which must be dependent on organism-species, and the linkage-specificity of sulfation not only  www.nature.com/scientificreports/ (TF) from humans was purchased from Wako (Osaka, Japan). TRI REAGENT LS was a product of Molecular Research Center, Inc. (Cincinnati, USA). pGEM-T Easy plasmid was purchased from Promega (Madison, WI, USA). The pcDNA3.1(neo) plasmid, pcDNA4-V5/His, and the mMessage mMachine SP6 transcription kits were purchased from Thermo Fisher Scientific KK (Tokyo, Japan). pSUPER.neo vector was a product of Oligoengine (Seattle, WA, USA). AmpliScribe T7-Flash Transcription kit was a product of Lucigen (WI, USA). ProtoScript II and BsaI were products of New England Biolabs (Ipswich, USA). The Cas9 expression vector with SP6 promoter, pCS2 + hSpCas9, and the sgRNA expression vector with a T7 promoter, pDR274, were gifts from Masato Kinoshita (Addgene 51815) 55 , and Keith Joung (Addgene 42250) 56  ) was prepared as previously described 45 . The monoclonal IgM antibody 3G9 (3G9), which specifically recognizes the α-glycosides of Neu5Ac8S, was previously prepared using sea urchin sperm as an immunogen 45,46 . mAb.2G9 (10 μg/mL) was obtained as an IgM clone, which did not react with Neu5Ac8S-containing gangliosides, during the 3G9 preparation and was used as the isotype control. Unless otherwise stated, primers for PCR amplification were purchased from RIKAKEN (Nagoya, Japan).  www.nature.com/scientificreports/ Fluorometric high performance liquid chromatography (HPLC) analysis. To assess the amount of SiaS components of the glycoproteins blotted onto PVDF membranes 57 , E14.5 mouse brain homogenates (100 µg) were applied to 10% SDS-PAGE followed by blotting on a PVDF membrane. The left edge of the membrane was removed and used for western blotting with 3G9. The rest of the membrane was equally cut into nine pieces according to the molecular size. Each slit was cut into small pieces and subjected to mild acid hydrolysis in 0.4 mL of 0.1 N TFA at 80 °C for 2 h. The hydrolysates were dried using SpeedVac vacuum concentrator (Savant, Thermo Fisher Scientific). Twenty microliters each of 0.01 M TFA and DMB solution 39,45 were added and incubated at 50 °C for 2 h. The DMB-derivatized samples were directly applied to an octadecylsilyl (ODS) column (250 × 4.6 mm i.d., Capcellpak C18 type MG, Shiseido, Tokyo, Japan) and eluted with acetonitrile/ methanol/0.05% TFA (4:6:90, v/v/v) at 1.0 mL/min for 120 min on a JASCO HPLC system (excitation, 373 nm; emission, 448 nm) as previously described 45 . 4MU-Neu5Ac8S was used as a positive control. For identification of DMB-derivatives of Neu5Ac and Neu5Ac8S, the identical retention times to the authentic sialic acid species were confirmed. In addition, co-injection experiments of the samples and CMP-Neu5Ac or CMP-Neu5Ac8S were also performed.

Ethics statement and the ARRIVE guidelines.
Cloning of Wscd1 and Wscd2 cDNA from mice and medaka. The cDNAs for the mouse cell wall integrity and stress response component (WSC) domain containing 1 (mWscd1) and mWscd2 genes (Gene ID: 216881 and 320916, respectively) were obtained by amplifying the coding regions by PCR using specific primers (Table S1) and Ex Taq DNA polymerase according to the manufacturer's protocol. Total RNA was prepared from mouse embryonic brain (E14.5) using TRI REAGENT LS. First strand cDNA was synthesized using random hexamer primers from 1 μg of total RNA as the template using ProtoScript II reverse transcriptase. The PCR conditions were 30 cycles of a step program (94 °C for 1 min, 55 °C for 30 s, and 72 °C for 1 min). The product was cloned into the pGEM-T Easy plasmid. DNA sequences were analyzed using the deoxynucleotide chain termination method. The cDNAs for medaka Wscd1 (mdkWscd1) and mdkWscd2 genes (gene ID: 101157150 and 101164728, respectively) were obtained by amplifying the coding regions through PCR using the primary cDNA prepared from 6-dpf and 7-dpf fry, respectively, primers (Table S1), and Ex Taq DNA polymerase. The PCR products were also cloned into the pGEM-T easy plasmid.

Molecular phylogenetic analysis.
Nucleotide sequences of genes that are annotated as mouse sulfotransferases or Wscd1/Wscd2 of various animals were obtained from the National Center for Biotechnology Information (NCBI) gene database (https:// www. ncbi. nlm. nih. gov/ gene/). Multiple sequence alignment of all the sequences was performed by ClustalW 2.1 (DNA Matrix; IUB, Slow Pairwise Alignment, Tossgaps) and the phylogenic tree was obtained by the Neighbor-joining method (Kimura method) on GENETYX software Ver.14.
Plasmid preparations. (a) Mammalian expression plasmids for Wscd1 and Wscd2. The cDNA fragments were amplified by a two-step cycle PCR (98 °C for 10 s, 68 °C for 1 min, 30 cycles) with KOD-Plus-Neo polymerase from the mWscd1-and mWscd2-encoded pGEM-T easy plasmids using the primers with an additional 15 bp at both 5′-and 3′-ends that matched the linearized pcDNA3.1-V5/His plasmid (Table S2), and subcloned into the pcDNA3.1-V5/His using an In-Fusion HD Cloning Kit. The mdkWscd1-, and mdkWscd2-encoded pcDNA plasmids were prepared by the same procedures, except that the corresponding pGEM-T easy plasmids and primers (Table S2), and pcDNA4-Myc/His plasmid were used. The obtained plasmids were denoted pcDNA-mWscd1, pcDNA-mWscd2, pcDNA-mdkWscd1, and pcDNA-mdkWscd2, respectively. The integrity of all plasmids was confirmed by DNA sequencing using the dideoxynucleotide chain termination method. (b) Plasmids for mutWscd1 and mutWscd2: To obtain an inactivated form of mWscd1 or mWscd2, and mutWscd1 or mutWscd2, the PAPS binding domain sequence (357-363 or 356-362 amino acids, respectively) was impaired (Supp_FigS5). Four critical amino acid residues (proline-357 or 356, glycine-360 or 359, threonine-362 or 361, and tryptophan-363 or 362 for mutWscd1 or mutWscd2, respectively) in the conserved PAPS binding domain were replaced with alanine residues. A sequential site-directed mutations of pcDNA-mWscd1 or pcDNA-mWscd2 (see above) were performed to obtain plasmids containing the four-amino acid-mutated mWscd1 or mWscd2 genes, respectively. The PCR conditions, templates, and primers used are described in Table S3. In each procedure, two-step cycle PCR (98 °C for 10 s, and 68 °C for 4 min; 30 cycles) was performed. The product was digested with DpnI to remove the remaining template plasmid. DH5⍺ cells were transformed with the DpnI-digested product (1 μL). The integrity of the plasmid was confirmed by DNA sequencing. (c) Short hairpin RNA (shRNA) plasmids: The shRNA plasmids were prepared using the pSUPER.neo vector according to the manufacturer's instructions. Sense and antisense oligonucleotides for suppressing the expression of human Wscd1 or Wscd2 gene (ID: 23302 or ID: 9671, respectively) were designed using the siDirect tool (http:// sidir ect2. rnai. jp), and purchased from Eurofins Japan (Luxembourg, Netherlands) (Table S4). They were heated at 94 °C for 4 min in 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, annealed by slowly cooling to 20 °C in steps of 2 °C every 4 min, and ligated into the pSUPER.neo vector by T4 DNA ligase to obtain the shWscd1 and shWscd2 shRNA plasmids. As a control, the pSUPER.neo plasmid was used as shMock instead of shWscd1 or shWscd2. DH5⍺ cells were transformed with the Bgl II-digested product (1 μL). The integrity of the plasmid was confirmed by DNA sequencing. (d) pDR274 plasmid with sgWscd1 or sgWscd2 sequence: Construction of the pDR274 plasmids encoding the CRISPR-Cas9 targets was previously described 58 . Briefly, target sequences in sgRNAs for medaka Wscd1 or Wscd2 gene were designed using their sequences, ENSORLG00000000526 and ENSORLG00000006858, respectively, at the Ensembl Genome Database Project. Oligonucleotide pairs containing the target sequences (Table S5)  www.nature.com/scientificreports/ BsaI-digested pDR274 vector. The obtained plasmids were denoted pDR274-sgWscd1 and pDR274-sgWscd, respectively.
Cell culture. Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells were purchased from Riken Cell Bank (Tsukuba, Japan). The human neuroblastoma (SK-N-SH) cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (Kobe, Japan). CHO and SK-N-SH cells were cultured in MEM-α (Wako, Japan) supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate, and 10% fetal bovine serum in a 5% CO 2 and 95% air-humidified atmosphere at 37 °C. HEK cells were cultured under the same conditions except that Dulbecco's modified Eagle's medium was used instead of MEM-α.
Cell transfection. CHO cells (0.5 × 10 6 cells) were cultured in a 6-well plate overnight at 37 °C, and transiently transfected with 3 μg of each pcDNA3.1 plasmid for mWscd1 and mWscd2, and pcDNA4 plasmids for mdkWscd1 and mdkWscd2 using the PEI-Max Transfection Reagent. At 48 h post-transfection, the cells were collected and subjected to flow cytometry and fluorometric HPLC analyses to evaluate the SiaS epitope. Transfection efficiency was evaluated by observing the transfected cells with a plasmid encoding the green fluorescent protein (GFP) gene by fluorescent microscopy. For RNA interference with shRNA plasmids, transfection of HEK and SK-N-SH cells was performed with the shWscd1 and shWscd2 plasmids, respectively, according to the above-mentioned method. , v/v/v). The TLC sheet was cut into two parts. One part was visualized for detecting the acidic glycolipid products (P) by the orcinol/sulfuric acid method 33,59 . The other part was used to collect the product P by scratching the silica gels at the corresponding position of P on the visualized plate. The product P was extracted from the collected silica gels by CMW, and the supernatant was subjected to fluorometric HPLC after hydrolyzed in 0.02 mL of 0.1 N TFA at 80 °C for 2 h. (c) TF substrate: The reaction mixtures with and without TF were subjected to SDS-PAGE/western blotting with 3G9 as described above. CBB staining was performed to check for equal substrate loading in the western blotting. The reaction products were also analyzed by the fluorometric HPLC, after hydrolyzed in 0.1 N TFA.

Flow cytometry analysis (FCA
Quantitative RT-PCR (qPCR). Total RNA was extracted from cells and tissues by using TRI REAGENT LS. The amount of extracted RNA was quantified using a spectrophotometer, and the purity of RNA was checked by the ratio of absorbance at 260 nm and 280 nm. Total RNA (5 μg) was subjected to reverse transcription using ProtoScript II with a random hexamer primer. qRT-PCR was performed using a pair of oligonucleotides (Table S6) and SYBR GreenER qPCR Supermix for iCycler premix. Amplification was performed using the iCycler IQ real-time PCR analysis system (Bio-Rad). Gene expression profile modulations were assessed by www.nature.com/scientificreports/ comparing the Ct values using the 2 −∆∆Ct method. The medaka actin gene was used to normalize the expression of the genes of interest. All experiments were conducted in triplicate.
Generation of Wscd1-and Wscd2-deficient medaka. All the procedures followed the instruction protocol by the NIBB55 (https:// shigen. nig. ac. jp/ medaka/) 58 . After pCS2 + hSpCas9 was linearized by NotI digestion, the vector was used as a template for the synthesis of capped Cas9 mRNA with an mMessage mMachine SP6 kit and then purified using the RNeasy Mini Kit. For the synthesis of sgRNAs, the pDR274 vector containing sgWscd1 or sgWscd2 was first digested by DraI. This was use as the template for synthesizing sgRNA using the AmpliScribe T7-Flash Transcription kit. The sgRNAs were purified using the RNeasy Mini kit. Approximately 2-4 nL of a mixture containing 100 ng/µL of Cas9 mRNA and 25 ng/µL of sgRNA of Wscd1 or Wscd2 was injected into embryos at the one-cell stage.
Genotyping. The fin clips of selected medaka fish or larvae were fixed in 40 μL of methanol and lysed in an appropriate amount of protease K solution (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, and 2 mg/mL proteinase K). It was then incubated at 55 °C for 3 h, followed by denaturation at 95 °C for 15 min to inactivate the protease K. After centrifugation, the supernatant of each sample was used as genomic DNA. To detect CRISPR/ Cas9-induced mutations, a heteroduplex mobility assay (HMA) was performed as described previously [60][61][62] . The mutations were then sequenced using an appropriate primer set (Table S7).
Assessment of survival rate of medaka. The G0 medaka were out-crossed with the wild-type medaka to obtain the F1 hetero-mutant medaka. The F1 hetero-mutants were subjected to genotyping to find Wscd1(+/−) and Wscd2(+/−) medaka, which contained a knockout allele arisen from frameshifts. The Wscd1(+/−) or Wscd2(+/−) female and male medaka of the same genotype were in-crossed with each other to obtain Wscd1(−/−) or Wscd2(−/−) offspring at F2. The Wscd1(+/−) or Wscd2(+/−) female and male medaka were carefully maintained as the stable strain containing the knockout allele. Their offspring that should contain all the (−/−), (+/−) and (+/+) genotypes were daily collected and maintained in a separate plastic rearing tank under a 14 h-day/10 h-night cycle at 26 °C. For each group, at least 5 small tanks were established to assess the life span. To understand the life span, the medaka were observed every day for a certain period. When some of them died, the dead fish were immediately collected for genotyping.
Digital video recording and analysis of the heart contraction. Young fry from 3 to 9 dpf (hatching day) was immobilized in a hole made by 1.5% agarose. The heart contractions were recorded using an SZX12 DP80 microscope (Olympus). Digital pictures were captured at maximum frame rate at a resolution of 1360-1024 pixels for up to 3 min and recorded in a PC using CellSens Standard software. Movies of heart movements were processed using ImageJ software. Contraction rhythms were measured based on alterations in the intensity of blood cell flow into and out of ventricle. Regions of interest (ROIs) in the ventricle were selected. The pixel intensities of the ROIs were digitized throughout the entire time series examined using ImageJ software.
Growth assessment of WT and Wscd2(−/−). Young fry of Wscd2(−/−) that were alive at 60 dpf and WT fry were measured for the body weight and body length as described previously 63,64 . Briefly, after anesthetized, the fish were weighed, and the body length was measured using the ruler. Five Wscd2(−/−) fish and 15 WT fish were used.
Statistics. All values were expressed as the mean SE (n is three) and p-values were evaluated by the Student's t-test.

Data availability
The nucleotide sequences reported in this paper will appear in the DNA Data Bank Japan (DDBJ) nucleotide sequence databases with LC669910 for mouse Wscd2; LC669911 for medaka Wscd1; and LC669912 for medaka Wscd2. Enter the ID at the DDBJ site: http:// geten try. ddbj. nig. ac. jp/ top-e. html.